GSM7977072: TERRA_captured_Nanopore_direct_RNA-seq; Homo sapiens; RNA-Seq
Total RNA was extracted from 1 x 10^7 cells using TRIzol™ Reagent (Invitrogen™, Cat:15596018) following the manufacturer's protocol. For Illumina sequencing, the cDNA library was constructed using the NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, E7760S) according to the manufactory instruction with some modifications. The first-strand synthesis step was carried out by SuperScript IV (Thermo Fisher Scientific, Cat: 18090010). The TERRA-captured RNA sample was divided into two equal halves for the reverse transcription reaction. Half of the TERRA-captured sample was reverse transcribed using 2 μM random hexamers (Thermo Fisher Scientific, 48190011), while the other half using 1 μM of telomeric C-rich primer. These two groups are referred to as the random and specific groups, respectively. The rest steps were performed as the protocol provided by manufactor. For Nanopore sequencing, TERRA-enriched sample was polyadenylated using E. coli Poly (A) polymerase (New England Biolabs, #M0276S) at a final concentration of 0.25 U/μl in 1X Poly(A) Polymerase Reaction Buffer with 1 mM dATP and 2 Unit/μl RNaseOUT, (Thermo Fisher Scientific, Cat: 10777019) at 37°C for 30 minutes. The reaction was terminated by adding 10 mM EDTA. The RNA sample was purified by AMPureXP RNA beads (Beckman Coulter, A66514) then subjected to Nanopore direct RNA sequencing protocol (Cat#SQK-RNA002).